This invention is in the field of plant molecular biology. More specifically, this invention pertains to nucleic acid fragments encoding thiamine biosynthetic enzymes in plants and seeds.
Many vertebrates, including man, lack the ability to manufacture certain essential cofactors and therefore these cofactors must be made available and therefore must be present in their diet. These cofactors are vitamins. Plants serve as the ultimate source of vitamins for humans and animals, thus, the ability to manipulate the production and accumulation of vitamins in plants would be of considerable importance and value. Furthermore, the inability of humans and animals to synthesize these cofactors provides a useful distinction between human or animal and plant cellular metabolism. This distinction can be exploited for the discovery of herbicidal chemical compounds that target enzymes in the plant biosynthetic pathways of vitamins while having a low toxicity to animals.
Thiamine, or vitamin B1, is a cofactor for transketolase, pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase. The biosynthetic pathway for thiamine production has been extensively studied in yeasts and bacteria but little plant data is available. The E. coli thiamine biosynthetic enzyme C (thic) has a predicted molecular weight of 70 kDa (Vander Horn et al. (1993) J. Bacteriol. 175:982-992). The B. subtilis thiamine biosynthetic enzyme C has thiamine phosphate synthase activity converting thiazole phosphate and pyrimidine pyrophosphate to thiamine (Zhang et al. (1997) J. Bacteriol. 179:3030-3035). The THI4 gene from S. cerevisiae is induced upon the depletion of thiamine and appears to play a role in mitochondrial DNA damage control (Machado et al. (1997) J. Mol. Biol. 273:114-121). Genes encoding two corn thiamine biosynthetic enzymes (thi1-1 and thi1-2) which complement the yeast thi4 mutant have been identified. These cDNAs have very little similarity at their 3xe2x80x2 untranslated regions. The polypeptides encoded by these genes are involved in the production of thiazole, a thiamine precursor, have a conserved central region and more variable amino- and carboxy- terminii. Both polypeptides contain signal sequences which resemble plastid transit peptides (Belanger et al. (1995) Plant Mol. Biol. 29:809-821).
Identification of the genes encoding thi1-1, thi1-2, and thiC will allow the manipulation of vitamin B1 production in crop plants and thereby allowing for the improvement of their nutritional value. Manipulation of these enzymes may affect the achievable levels of xanthophylls in corn endosperm. Finally, these enzymes are involved in the production of plastid-derived isoprenoids, and as such are good herbicide targets.
The present invention concerns an isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of: (a) first a nucleotide sequence comprising at least 450 nucleotides selected from SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, and 43; (b) a second nucleotide sequence encoding a polypeptide of at least 90 amino acids having at least 97% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, and 44; and (c) a third nucleotide sequence comprising the complement of (a) or (b).
In a second embodiment, this invention relates to a chimeric gene comprising an isolated polynucleotide of the present invention operably linked to at least one suitable regulatory sequence.
In a third embodiment, the present invention concerns a host cell comprising a chimeric gene of the present invention or an isolated polynucleotide of the present invention. The host cell may be eukaryotic, such as a yeast or a plant cell, or prokaryotic, such as a bacterial cell. The present invention also relates to a virus, preferably a baculovirus, comprising an isolated polynucleotide of the present invention or a chimeric gene of the present invention.
In a fourth embodiment, the invention also relates to a process for producing a host cell comprising a chimeric gene of the present invention or an isolated polynucleotide of the present invention, the process comprising either transforming or transfecting a compatible host cell with a chimeric gene or isolated polynucleotide of the present invention.
In a fifth embodiment, the invention concerns a thi or a thiC polypeptide of at least 90 amino acids comprising at least 97% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, and 44.
In a sixth embodiment, the invention relates to a method of selecting an isolated polynucleotide that affects the level of expression of a thi1 or a thiC polypeptide or enzyme activity in a host cell, preferably a plant cell, the method comprising the steps of: (a) constructing an isolated polynucleotide of the present invention or a chimeric gene of the present invention; (b) introducing the isolated polynucleotide or the chimeric gene into a host cell; (c) measuring the level of the thi1 or the thiC polypeptide or enzyme activity in the host cell containing the isolated polynucleotide; and (d) comparing the level of the thi1 or the thiC polypeptide or enzyme activity in the host cell containing the isolated polynucleotide with the level of the thi or the thiC polypeptide or enzyme activity in the host cell that does not contain the isolated polynucleotide.
In a seventh embodiment, the invention concerns a method of obtaining a nucleic acid fragment encoding a substantial portion of a thi1 or a thiC polypeptide, preferably a plant thi1 or thiC polypeptide, comprising the steps of: synthesizing an oligonucleotide primer comprising a nucleotide sequence of at least 60 (preferably at least 40, most preferably at least 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, and 43, and the complement of such nucleotide sequences; and amplifying a nucleic acid fragment (preferably a cDNA inserted in a cloning vector) using the oligonucleotide primer. The amplified nucleic acid fragment preferably will encode a substantial portion of a thi1 or a thiC amino acid sequence.
In an eighth embodiment, this invention relates to a method of obtaining a nucleic acid fragment encoding all or a substantial portion of the amino acid sequence encoding a thi1 or a thiC polypeptide comprising the steps of: probing a cDNA or genomic library with an isolated polynucleotide of the present invention; identifying a DNA clone that hybridizes with an isolated polynucleotide of the present invention; isolating the identified DNA clone; and sequencing the cDNA or genomic fragment that comprises the isolated DNA clone.
In a ninth embodiment, this invention concerns a composition, such as a hybridization mixture, comprising an isolated polynucleotide or an isolated polypeptide of the present invention.
In a tenth embodiment, this invention concerns a method for positive selection of a transformed cell comprising: (a) transforming a host cell with the chimeric gene of the present invention or a construct of the present invention; and (b) growing the transformed host cell, preferably a plant cell, such as a monocot or a dicot, under conditions which allow expression of the thi1 or the thiC polynucleotide in an amount sufficient to complement a null mutant to provide a positive selection means.
In an eleventh embodiment, this invention relates to a method of altering the level of expression of a thiamine biosynthetic enzyme in a host cell comprising: (a) transforming a host cell with a chimeric gene of the present invention; and (b) growing the transformed host cell under conditions that are suitable for expression of the chimeric gene wherein expression of the chimeric gene results in production of altered levels of the thiamine biosynthetic enzyme in the transformed host cell.
In a twelfth embodiment, this invention concerns an isolated polynucleotide comprising a nucleotide sequence of at least 60 (preferably at least 40, most preferably at least 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, and 37, and the complement of such sequences.
A further embodiment of the instant invention is a method for evaluating at least one compound for its ability to inhibit the activity of a thiamine biosynthetic enzyme, the method comprising the steps of: (a) transforming a host cell with a chimeric gene comprising a nucleic acid fragment encoding the thiamine biosynthetic enzyme, operably linked to at least one suitable regulatory sequence; (b) growing the transformed host cell under conditions that are suitable for expression of the chimeric gene wherein expression of the chimeric gene results in production of the thiamine biosynthetic enzyme in the transformed host cell; (c) optionally purifying the thiamine biosynthetic enzyme expressed by the transformed host cell; (d) treating the thiamine biosynthetic enzyme with a compound to be tested; and (e) comparing the activity of the thiamine biosynthetic enzyme that has been treated with a test compound to the activity of an untreated thiamine biosynthetic enzyme, thereby selecting compounds with potential for inhibitory activity.